Recombinant Photobacterium profundum Probable transcriptional regulatory protein PBPRB1582 (PBPRB1582)

What is Photobacterium profundum PBPRB1582 and what is its role in bacterial adaptation?

PBPRB1582 is a probable transcriptional regulatory protein found in Photobacterium profundum, a deep-sea bacterium belonging to the Vibrionaceae family. As a transcriptional regulator, it likely plays a crucial role in the bacterium's adaptation to high-pressure environments. P. profundum is a gram-negative rod that can grow at temperatures from 0°C to 25°C and pressures from 0.1 MPa to 70 MPa, depending on the strain .

The strain SS9 specifically exhibits optimal growth at 15°C and 28 MPa, making it both a psychrophile and a piezophile . PBPRB1582's sequence (MGRKFEVRKLSMAKTAGAKIKVYSKYGKEIYVCAKNGSLDPDSNLSLKRLIEKAKKDQVPSHVIEKAIDKAKGGAGEEFATARYEWFGPRYCMVIVDCLTDNNNRTFMDVRQAFVKNHAKIGGPGTVGHMFEHQAVFQFAGDDEDMVLENLMMEDVDVSDIECEDGIITVYAPHTEFFKVKNALAATMPDVAFDVEEISFVPQTMTEISGDDVAAFEKFLDVLNDCDDVQNIYHNAEIAE) suggests DNA-binding capabilities essential for transcriptional control .

Research methodology for understanding PBPRB1582's role should include:

• Comparative genomic analysis against other deep-sea bacterial transcriptional regulators

• Expression profiling under varied pressure conditions

• DNA-binding assays to identify target promoter regions

• Creation of knockout mutants to observe phenotypic changes in pressure adaptation

How does high hydrostatic pressure affect transcriptional regulation in P. profundum?

High hydrostatic pressure significantly impacts transcriptional regulation in P. profundum, with specific regulatory proteins like ToxR showing pressure-dependent activity. RNA-seq analysis revealed a complex expression pattern with 22 genes having expression profiles similar to OmpH, an outer membrane protein transcribed in response to high hydrostatic pressure .

The pressure response transcriptome in P. profundum includes:

Transcriptional regulators like PBPRB1582 likely coordinate these pressure-specific adaptations, modulating gene expression to optimize cellular processes under deep-sea conditions . RNA-seq analysis has identified 460 putative small RNA genes and revealed 992 genes with large 5'-UTRs capable of harboring cis-regulatory RNA structures, suggesting complex transcriptional and post-transcriptional regulation mechanisms .

For experimental design, researchers should:

• Perform differential gene expression analysis comparing high pressure (28 MPa) vs. atmospheric pressure conditions

• Include time-course experiments to capture early, middle, and late adaptation responses

• Analyze both protein-coding and non-coding RNA expression changes

• Validate key findings with qRT-PCR and Western blotting

What methodologies are recommended for studying PBPRB1582 binding activity?

To study PBPRB1582 binding activity, researchers should employ multiple complementary approaches:

Chromatin Immunoprecipitation (ChIP) Analysis:

• Express PBPRB1582 with an epitope tag in P. profundum using conjugal delivery systems similar to those used for flagellar gene studies

• Cross-link protein-DNA complexes in vivo using formaldehyde treatment

• Immunoprecipitate PBPRB1582-bound DNA fragments using antibodies against the epitope tag

• Sequence precipitated DNA (ChIP-seq) to identify genome-wide binding sites

Electrophoretic Mobility Shift Assays (EMSA):

• Express and purify recombinant PBPRB1582 (>85% purity by SDS-PAGE)

• Generate labeled DNA probes from predicted binding regions

• Incubate protein with probes and analyze by native gel electrophoresis

• Include competition assays with unlabeled DNA to verify binding specificity

DNase I Footprinting:

• Prepare end-labeled DNA fragments containing putative binding sites

• Incubate with purified PBPRB1582 protein

• Perform limited DNase I digestion

• Analyze protected regions by sequencing gel electrophoresis

For all binding studies, it's critical to test activity under various pressure conditions using specialized high-pressure equipment similar to the HPDS high-pressure cell employed for motility studies , as PBPRB1582 activity may be pressure-dependent like other P. profundum regulatory systems.

How can researchers effectively express recombinant PBPRB1582 for functional studies?

The expression of recombinant PBPRB1582 requires careful consideration of expression systems, purification strategies, and validation of functional activity. Based on successful approaches with other P. profundum proteins:

Expression Systems:

• E. coli Expression: Use BL21(DE3) strain with pET vector systems, optimizing for cold-adapted protein expression (15-20°C induction temperature)

• Yeast Expression: Consider Pichia pastoris for proteins difficult to express in prokaryotic systems

• Cell-Free Expression: For pressure-sensitive proteins that may fold incorrectly in standard systems

Purification Protocol:

• Lyse cells in buffer containing appropriate salt concentration (mimicking marine conditions)

• Perform initial purification using affinity chromatography (His-tag or FLAG-tag)

• Further purify using ion exchange chromatography on Bio-Rex 70 cation exchange resin

• Final polishing step with size exclusion chromatography

• Verify purity (>85%) using SDS-PAGE

Storage Recommendations:

• Store purified protein at -20°C/-80°C with 50% glycerol as cryoprotectant

• Shelf life of liquid form: approximately 6 months at -20°C/-80°C

• Shelf life of lyophilized form: approximately 12 months at -20°C/-80°C

• Avoid repeated freezing and thawing; store working aliquots at 4°C for up to one week

Functional Validation:

Verify DNA-binding activity using gel shift assays and confirm pressure response characteristics using high-pressure experimental chambers to simulate native deep-sea conditions.

What is known about the genomic context and evolutionary conservation of PBPRB1582?

The genomic context of PBPRB1582 provides important insights into its evolutionary significance and functional relationships. While specific information about PBPRB1582's genomic neighborhood is limited in the provided sources, general principles can guide research in this area:

P. profundum strain SS9's genome consists of a 4.1-Mbp circular chromosome, a 2.2-Mbp minor circular chromosome, and an 80-kbp circular plasmid. The distribution of genes across these chromosomes is functionally significant - chromosome 1 contains genes essential for growth, while chromosome 2 appears to have evolved from a large plasmid and contains numerous transposable elements.

Researchers investigating PBPRB1582's genomic context should:

• Determine whether PBPRB1582 is located on chromosome 1 or 2, as this placement provides clues about its essentiality

• Identify neighboring genes that may form an operon structure

• Compare synteny with related Vibrionaceae species to understand evolutionary conservation

• Analyze promoter regions for binding sites of known pressure-responsive regulators like ToxR

Based on 16S rRNA sequence analysis, P. profundum is closely related to the genus Vibrio, particularly Vibrio cholerae . Comparative genomics between these species may reveal conservation patterns of transcriptional regulators like PBPRB1582, potentially identifying orthologs with similar functions in different pressure environments.

How does PBPRB1582 potentially interact with the ToxR regulatory system?

The interaction between PBPRB1582 and the ToxR regulatory system represents an important research direction, as ToxR is a well-characterized pressure-responsive regulator in P. profundum. ToxR is a transmembrane DNA-binding protein first discovered in Vibrio cholerae that regulates genes involved in environmental adaptation and virulence . In P. profundum, ToxR's abundance and activity are influenced by hydrostatic pressure, governing the regulation of genes in a pressure-dependent manner .

RNA-seq analysis comparing wild-type and toxR mutant strains revealed a complex expression pattern with 22 genes showing profiles similar to OmpH, an outer membrane protein transcribed in response to high hydrostatic pressure . As a probable transcriptional regulator, PBPRB1582 may:

• Function within the ToxR regulon as a downstream effector

• Act as a co-regulator working in concert with ToxR

• Regulate a parallel pathway that complements ToxR-mediated adaptations

• Compete with ToxR for binding to overlapping target sequences

To investigate these potential relationships, researchers should:

• Perform comparative transcriptomics between wild-type, toxR mutant, and PBPRB1582 mutant strains

• Utilize ChIP-seq to identify potential overlap in ToxR and PBPRB1582 binding sites

• Conduct bacterial two-hybrid or co-immunoprecipitation experiments to detect physical interactions

• Engineer strains with inducible expression of each regulator to test epistatic relationships

Understanding this regulatory network is crucial for deciphering how P. profundum coordinates its complex adaptation to deep-sea environments.

What experimental approaches are recommended for studying PBPRB1582 function under high-pressure conditions?

Investigating PBPRB1582 function under high-pressure conditions requires specialized equipment and methodological approaches:

High-Pressure Cultivation Systems:

• Use high-pressure vessels capable of maintaining 28 MPa (optimal for P. profundum SS9)

• Implement temperature control systems to maintain 15°C during experiments

• Consider the HPDS high-pressure cell system used for visualizing motility under pressure

Gene Expression Analysis Under Pressure:

• Perform RNA extraction immediately after pressure release to capture authentic expression profiles

• Use RT-PCR with controls such as uridine phosphorylase (udp; PBPRA1431) for normalization

• Consider RNA-seq for genome-wide expression analysis under varying pressure conditions

Mutant Construction and Complementation:

• Employ marker exchange-eviction mutagenesis using the sacB-containing suicide vector pRL271

• Prepare deletion constructs by PCR amplification of upstream and downstream regions

• Verify mutants by PCR and phenotypic analysis

• Complement with wild-type gene using vectors like pFL122 for functional validation

Pressure-Shift Experiments:

Design experimental protocols that include:

• Baseline gene expression at atmospheric pressure (0.1 MPa)

• Short-term pressure shifts (minutes to hours) to capture immediate regulatory responses

• Long-term adaptation studies (days) to identify stable regulatory networks

• Combined pressure and temperature shifts to understand interactive environmental effects

This approach mirrors successful methodologies used to study other pressure-responsive systems in P. profundum, such as the flagellar genes and ToxR regulon .

How can researchers resolve contradictory data when studying PBPRB1582 regulation?

Resolving contradictory data is a common challenge in research on deep-sea bacteria due to the complexity of pressure responses and technical limitations. When studying PBPRB1582 regulation, researchers may encounter apparently conflicting results that require careful analysis:

Common Sources of Contradictions:

• Strain-specific differences: Various P. profundum strains (SS9, 3TCK, DJS4, 1230) have different pressure optima

• Experimental pressure conditions: Acute vs. chronic pressure exposure produces different responses

• Temperature interactions: Pressure effects are often temperature-dependent

• Growth phase variations: Exponential vs. stationary phase cells show different regulatory patterns

• Technical artifacts from decompression: RNA/protein extraction after pressure release may alter profiles

Resolution Strategies:

• Standardize experimental conditions:

  • Use defined growth media and consistent cell densities (OD₆₀₀ = 0.3 for mid-exponential phase)

  • Maintain consistent pressure application/release rates

  • Control temperature precisely at 15°C for SS9 strain studies

• Employ multiple methodologies:

  • Complement transcriptomic data with proteomic analysis

  • Validate RNA-seq findings with targeted qRT-PCR

  • Verify in vitro binding studies with in vivo reporter assays

• Consider temporal dynamics:

  • Sample at multiple time points after pressure shifts

  • Differentiate between immediate stress responses and adaptive regulation

• Genetic approaches:

  • Create clean deletion mutants of PBPRB1582 using marker exchange-eviction methods

  • Perform complementation studies with wild-type and mutant versions

  • Conduct epistasis analysis with related regulators like ToxR

When presenting seemingly contradictory findings, organize data in tables showing the specific conditions under which each result was obtained, enabling more systematic comparison and identification of pattern-explaining variables.

What is the relationship between PBPRB1582 and adaptation to deep-sea conditions beyond pressure?

While hydrostatic pressure is a defining feature of deep-sea environments, PBPRB1582 likely participates in broader adaptive responses to the deep-sea milieu, which includes low temperature, limited nutrients, and other unique physicochemical properties:

Temperature Adaptation:

P. profundum SS9 is both a piezophile and a psychrophile, with optimal growth at 15°C . PBPRB1582 may coordinate transcriptional responses that link pressure and cold adaptation. At low temperatures and high pressure, strain SS9 increases the abundance of mono- and polyunsaturated fatty acids in its cell membrane, enhancing membrane fluidity . Transcriptional regulators like PBPRB1582 potentially govern the expression of genes involved in these membrane modifications.

Nutrient Acquisition and Metabolism:

Deep-sea environments represent distinct ecosystems with particular nutrient limitations and abundances . Proteomic analysis revealed that proteins involved in nutrient transport or assimilation are likely directly regulated by pressure . Research should investigate whether PBPRB1582 controls genes involved in:

• Specific carbon source utilization under pressure

• Nitrogen or phosphorus scavenging in nutrient-limited conditions

• Metabolic pathway switching between glycolysis and oxidative phosphorylation

Polyunsaturated Fatty Acid (PUFA) Biosynthesis:

P. profundum possesses pfa genes involved in PUFA biosynthesis, which are important for adaptation to cold, high-pressure environments . Investigating whether PBPRB1582 regulates these genes would provide insight into its role in coordinating multiple aspects of deep-sea adaptation.

Flagellar Regulation:

P. profundum SS9 utilizes separate flagellar systems for swimming and swarming under high-pressure conditions . These systems show pressure-dependent expression patterns, with lateral flagellar genes (including flaB and motA1) upregulated under high-pressure and high-viscosity conditions . Researchers should determine if PBPRB1582 participates in this specialized motility regulation.

How can RNA-seq methodology be optimized for studying PBPRB1582 regulatory networks?

RNA-seq provides powerful insights into transcriptional regulatory networks but requires optimization for studying pressure-responsive systems like those potentially controlled by PBPRB1582:

Experimental Design Considerations:

• Pressure Conditions:

  • Compare multiple pressure points (0.1 MPa, 10 MPa, 28 MPa, 40 MPa)

  • Include time-course sampling to capture dynamic responses

  • Control temperature consistently at 15°C (optimal for SS9)

• Strain Selection:

  • Include wild-type SS9 and PBPRB1582 deletion mutant

  • Consider including ToxR mutant (TW30) for comparative analysis

  • Use additional control strains with mutations in related regulatory systems

• RNA Extraction Protocol:

  • Extract RNA immediately after pressure release to minimize artifacts

  • Use methods previously validated for P. profundum (as used in ToxR studies)

  • Include controls for RNA quality and degradation during decompression

Advanced Analysis Approaches:

• Genome-wide Prediction of Regulatory Features:

  • Analyze operon structure as previously demonstrated in P. profundum

  • Identify transcription start and termination sites

  • Map 5'-UTRs, particularly the large UTRs (992 genes) that may harbor cis-regulatory structures

• Differential Expression Analysis:

  • Use appropriate statistical methods accounting for biological replicates

  • Apply false discovery rate correction for multiple testing

  • Organize genes into co-regulated modules using clustering approaches

• Integration with Other Data Types:

  • Combine with ChIP-seq data to distinguish direct vs. indirect regulation

  • Correlate with proteomic data to account for post-transcriptional regulation

  • Incorporate metabolomic data to link transcriptional changes to phenotypic outcomes

This approach builds upon the RNA-seq methodology successfully employed to characterize the ToxR regulon in P. profundum , which identified 460 putative small RNA genes and detected 298 previously unknown protein-coding genes.

What are the recommended controls for experiments involving recombinant PBPRB1582?

Properly designed controls are essential for experiments with recombinant PBPRB1582 to ensure reliable and interpretable results:

Protein Expression and Purification Controls:

• Expression System Controls:

  • Empty vector control expressing tag only

  • Unrelated protein with similar size/properties and same tag

  • Known functional transcription factor from P. profundum with same tag

• Protein Quality Controls:

  • SDS-PAGE with Coomassie staining to verify >85% purity

  • Western blot with tag-specific antibody

  • Mass spectrometry validation of intact protein

  • Circular dichroism to confirm proper folding

Functional Assay Controls:

• DNA-Binding Assays:

  • Non-specific DNA sequences (scrambled or from unrelated organisms)

  • Mutated binding site versions to identify critical nucleotides

  • Competition assays with unlabeled DNA

  • Heat-denatured PBPRB1582 as negative control

• Transcriptional Regulation Assays:

  • Reporter constructs lacking promoter elements

  • Reporters with known ToxR-dependent promoters as comparative controls

  • Dose-response series with varying PBPRB1582 concentrations

  • Assays under varying pressure conditions (0.1 MPa vs. 28 MPa)

Genetic Complementation Controls:

• For in vivo Studies:

  • Empty vector control

  • Vector expressing PBPRB1582 with inactivating mutations

  • Vector expressing related transcriptional regulator

  • Wild-type strain (positive control)

• Pressure Response Controls:

  • Assays at atmospheric pressure (negative control for pressure-dependent activity)

  • ToxR-dependent system as positive control for pressure response

  • Temperature controls (conduct experiments at different temperatures)

These controls should be systematically incorporated into experimental designs, following approaches similar to those used in studying other pressure-responsive systems in P. profundum, such as the ToxR regulon and flagellar gene expression .

How can bioinformatic approaches help predict PBPRB1582 binding sites and regulatory targets?

Bioinformatic approaches provide powerful tools for predicting PBPRB1582 binding sites and regulatory targets, helping to guide experimental verification:

Motif Discovery and Binding Site Prediction:

• De Novo Motif Discovery:

  • Use algorithms like MEME, STREME, or HOMER to identify enriched sequence patterns near differentially expressed genes

  • Perform analysis on promoter regions of genes co-regulated under high pressure

  • Compare motifs with known transcription factor binding sites in related bacteria

• Phylogenetic Footprinting:

  • Identify conserved non-coding sequences in orthologous genes across Vibrionaceae

  • Focus on regulatory regions of pressure-responsive genes identified in RNA-seq studies

  • Weight conservation patterns by phylogenetic distance

• Structural Prediction:

  • Generate homology models of PBPRB1582 DNA-binding domain

  • Perform DNA-protein docking simulations to predict binding specificity

  • Use molecular dynamics simulations to assess pressure effects on binding

Regulon Prediction and Network Analysis:

• Co-expression Network Analysis:

  • Construct gene networks from RNA-seq data under various pressure conditions

  • Identify gene clusters with similar expression patterns

  • Integrate with ToxR regulon data to find overlaps and unique targets

• Comparative Genomics Approach:

  • Analyze gene neighborhood conservation across piezophilic bacteria

  • Identify conserved gene pairs or operons that may be co-regulated

  • Compare with known pressure-responsive genes in related organisms

• Functional Enrichment Analysis:

  • Perform GO term and pathway enrichment on predicted regulon members

  • Look for enrichment of specific metabolic pathways or stress responses

  • Connect to known pressure adaptation mechanisms like PUFA biosynthesis

Implementation Strategy:

• Begin with whole-genome scanning for probable binding sites based on consensus motifs from related transcriptional regulators

• Narrow candidates by focusing on promoters of pressure-responsive genes

• Prioritize targets with multiple lines of evidence (motif match, co-expression, conservation)

• Validate top predictions with experimental approaches like ChIP-seq and reporter assays

This strategy builds upon bioinformatic approaches successfully used for other bacterial transcriptional regulators while incorporating the specific context of deep-sea bacterial adaptations.